Bottom Line:
Deletion of ergM resulted in severe growth defects with increased sensitivity to azole-type antifungal agents but not amphotericin B.The data defined gene classes based on spatio-temporal expression such as those expressed early in the dimorphic switch but not in the terminal cell types and those expressed late.Such classifications have been helpful in linking a given gene of interest to its expression pattern throughout the P. marneffei dimorphic life cycle and its likely role in pathogenicity.

Affiliation: Department of Genetics, University of Melbourne, Victoria 3010, Australia.

ABSTRACTPenicillium marneffei is an opportunistic human pathogen endemic to Southeast Asia. At 25° P. marneffei grows in a filamentous hyphal form and can undergo asexual development (conidiation) to produce spores (conidia), the infectious agent. At 37° P. marneffei grows in the pathogenic yeast cell form that replicates by fission. Switching between these growth forms, known as dimorphic switching, is dependent on temperature. To understand the process of dimorphic switching and the physiological capacity of the different cell types, two microarray-based profiling experiments covering approximately 42% of the genome were performed. The first experiment compared cells from the hyphal, yeast, and conidiation phases to identify "phase or cell-state-specific" gene expression. The second experiment examined gene expression during the dimorphic switch from one morphological state to another. The data identified a variety of differentially expressed genes that have been organized into metabolic clusters based on predicted function and expression patterns. In particular, C-14 sterol reductase-encoding gene ergM of the ergosterol biosynthesis pathway showed high-level expression throughout yeast morphogenesis compared to hyphal. Deletion of ergM resulted in severe growth defects with increased sensitivity to azole-type antifungal agents but not amphotericin B. The data defined gene classes based on spatio-temporal expression such as those expressed early in the dimorphic switch but not in the terminal cell types and those expressed late. Such classifications have been helpful in linking a given gene of interest to its expression pattern throughout the P. marneffei dimorphic life cycle and its likely role in pathogenicity.

fig9: Growth rate defect of the ΔergM strain. (A) Radial growth rate (colony diameter) was measured every 24 hr for the control (ergM+), deletion (ΔergM), and complemented (ΔergM ergM+) strains at 25° on ANM medium. (B) Colony morphology after 4 d of growth on BHI medium at 25° and 37°.

Mentions:
To assess the role of ergM during growth and morphogenesis in P. marneffei, the ergM coding sequence was deleted by homologous gene replacement (see Materials and Methods section). Deletion of ergM led to significant and pleiotropic effects, including swollen and highly branched cells, slow growth, delayed conidiation, and altered antifungal sensitivity. The most obvious of these was a severe reduction in growth rate and altered colony morphology at both 25° and 37° (Figure 9). Microscopic analysis showed normal levels of progression past the germling stage but severe polarity defects in a proportion of cells at 25° (Figure 10), whereas at 37° a large proportion of the conidia failed to produce more than a small germ tube. The remaining cells grew at a reduced rate with very few yeast cells produced (Figure 10). The more severe phenotype at 37° confirmed the importance of ergM at this temperature. The growth reduction of the ergM deletion strain at both temperatures was initially severe; however, there was a marked recovery in growth over time (Figure 10). This recovery was not attributable to the accumulation of suppressor mutations because conidia from the deletion strain exhibited the severe growth phenotype of the original transformant at both temperatures (data not shown). The phenotypic change was therefore a biochemical or regulatory response to the altered sterol production. This phenomenon might be akin to a response observed in S. cerevisiae, in which other membrane components such as phospholipids were altered in ergosterol biosynthesis mutants, allowing growth (Sharma 2006).

fig9: Growth rate defect of the ΔergM strain. (A) Radial growth rate (colony diameter) was measured every 24 hr for the control (ergM+), deletion (ΔergM), and complemented (ΔergM ergM+) strains at 25° on ANM medium. (B) Colony morphology after 4 d of growth on BHI medium at 25° and 37°.

Mentions:
To assess the role of ergM during growth and morphogenesis in P. marneffei, the ergM coding sequence was deleted by homologous gene replacement (see Materials and Methods section). Deletion of ergM led to significant and pleiotropic effects, including swollen and highly branched cells, slow growth, delayed conidiation, and altered antifungal sensitivity. The most obvious of these was a severe reduction in growth rate and altered colony morphology at both 25° and 37° (Figure 9). Microscopic analysis showed normal levels of progression past the germling stage but severe polarity defects in a proportion of cells at 25° (Figure 10), whereas at 37° a large proportion of the conidia failed to produce more than a small germ tube. The remaining cells grew at a reduced rate with very few yeast cells produced (Figure 10). The more severe phenotype at 37° confirmed the importance of ergM at this temperature. The growth reduction of the ergM deletion strain at both temperatures was initially severe; however, there was a marked recovery in growth over time (Figure 10). This recovery was not attributable to the accumulation of suppressor mutations because conidia from the deletion strain exhibited the severe growth phenotype of the original transformant at both temperatures (data not shown). The phenotypic change was therefore a biochemical or regulatory response to the altered sterol production. This phenomenon might be akin to a response observed in S. cerevisiae, in which other membrane components such as phospholipids were altered in ergosterol biosynthesis mutants, allowing growth (Sharma 2006).

Bottom Line:
Deletion of ergM resulted in severe growth defects with increased sensitivity to azole-type antifungal agents but not amphotericin B.The data defined gene classes based on spatio-temporal expression such as those expressed early in the dimorphic switch but not in the terminal cell types and those expressed late.Such classifications have been helpful in linking a given gene of interest to its expression pattern throughout the P. marneffei dimorphic life cycle and its likely role in pathogenicity.

Affiliation:
Department of Genetics, University of Melbourne, Victoria 3010, Australia.

ABSTRACTPenicillium marneffei is an opportunistic human pathogen endemic to Southeast Asia. At 25° P. marneffei grows in a filamentous hyphal form and can undergo asexual development (conidiation) to produce spores (conidia), the infectious agent. At 37° P. marneffei grows in the pathogenic yeast cell form that replicates by fission. Switching between these growth forms, known as dimorphic switching, is dependent on temperature. To understand the process of dimorphic switching and the physiological capacity of the different cell types, two microarray-based profiling experiments covering approximately 42% of the genome were performed. The first experiment compared cells from the hyphal, yeast, and conidiation phases to identify "phase or cell-state-specific" gene expression. The second experiment examined gene expression during the dimorphic switch from one morphological state to another. The data identified a variety of differentially expressed genes that have been organized into metabolic clusters based on predicted function and expression patterns. In particular, C-14 sterol reductase-encoding gene ergM of the ergosterol biosynthesis pathway showed high-level expression throughout yeast morphogenesis compared to hyphal. Deletion of ergM resulted in severe growth defects with increased sensitivity to azole-type antifungal agents but not amphotericin B. The data defined gene classes based on spatio-temporal expression such as those expressed early in the dimorphic switch but not in the terminal cell types and those expressed late. Such classifications have been helpful in linking a given gene of interest to its expression pattern throughout the P. marneffei dimorphic life cycle and its likely role in pathogenicity.